Functional characterization and modified rescue of novel AE1 mutation R730C associated with overhydrated cation leak stomatocytosis

Abstract

SL. Functional characterization and modified rescue of novel AE1 mutation R730C associated with overhydrated cation leak stomatocytosis. Am J Physiol Cell Physiol 300: C1034 -C1046, 2011. First published January 5, 2011; doi:10.1152/ajpcell.00447.2010.-We report the novel, heterozygous AE1 mutation R730C associated with dominant, overhydrated, cation leak stomatocytosis and well-compensated anemia. Parallel elevations of red blood cell cation leak and ouabain-sensitive Na ϩ efflux (pump activity) were apparently unaccompanied by increased erythroid cation channel-like activity, and defined ouabain-insensitive Na ϩ efflux pathways of nystatin-treated cells were reduced. Epitope-tagged AE1 R730C at the Xenopus laevis oocyte surface exhibited severely reduced Cl Ϫ transport insensitive to rescue by glycophorin A (GPA) coexpression or by methanethiosulfonate (MTS) treatment. AE1 mutant R730K preserved Cl Ϫ transport activity, but R730 substitution with I, E, or H inactivated Cl Ϫ transport. AE1 R730C expression substantially increased endogenous oocyte Na ϩ -K ϩ -ATPase-mediated 86 Rb ϩ influx, but ouabain-insensitive flux was minimally increased and GPA-insensitive. The reduced AE1 R730C-mediated sulfate influx did not exhibit the wild-type pattern of stimulation by acidic extracellular pH (pH o) and, unexpectedly, was partially rescued by exposure to sodium 2-sulfonatoethyl methanethiosulfonate (MTSES) but not to 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA) or 2-(trimethylammonium)ethyl methanethiosulfonate bromide (MTSET). AE1 R730E correspondingly exhibited acid pH o-stimulated sulfate uptake at rates exceeding those of wild-type AE1 and AE1 R730K, whereas mutants R730I and R730H were inactive and pH o insensitive. MTSEStreated oocytes expressing AE1 R730C and untreated oocytes expressing AE1 R730E also exhibited unprecedented stimulation of Cl Ϫ influx by acid pH o. Thus recombinant cation-leak stomatocytosis mutant AE1 R730C exhibits severely reduced anion transport unaccompanied by increased Rb ϩ and Li ϩ influxes. Selective rescue of acid pHo-stimulated sulfate uptake and conferral of acid pH o-stimulated Cl Ϫ influx, by AE1 R730E and MTSES-treated R730C, define residue R730 as critical to selectivity and regulation of anion transport by AE1. cation leak; Xenopus oocyte; patch clamp; sulfate transport HEREDITARY STOMATOCYTOSIS (HSt) is characterized by overt or compensated, autosomal dominant anemia associated with increased red blood cell (RBC) cation leak insensitive to ouabain. The cation leaks of HSt RBCs from different cohorts exhibit widely varying temperature sensitivities and varied red cell membrane abundance of stomatin (Band 7.2) (11, 41), but red cells from all patients share elevated Na content and reduced K content. HSt with red cell cation leak (4) has been associated with multiple missense mutations in the AE1/

Rapid Cl−/HCO3− exchange kinetics of AE1 in HEK293 cells and hereditary stomatocytosis red blood cells

“…Human AE1 has already been stably expressed in HEK293 cells, using muristerone A induction on cells constitutively expressing ecdysone and retinoic acid receptors (30 (29), while membrane expression of R730C mutant was not (28). These results fit to our data in the HEK293 kidney cell line lacking erythroid proteins such as GPA, since E758K mutant was expressed in significant lesser amounts than R730C mutant (Newman-Keuls multiple comparison test, P Ͻ 0.05).…”

“…Indeed, although the alkalinization rate constant was five to six times lower for E758K mutant than for WT AE1 (0.07 vs. 0.38 s Ϫ1 ), normalization of transport values with surface expression level indicated comparable relative activities (98 vs. 100%) and apparent unit permeabilities (11.96 ϫ 10 Ϫ3 vs. 9.81 ϫ 10 Ϫ3 m 3 /s) of both E758K mutant and normal proteins. The defect in Cl Ϫ /HCO 3 Ϫ transport activity of G796R and R730C mutants was not due to reduced expression or processing of the proteins to the cell surface and also corroborates their inactivity reported in Xenopus oocytes (4,15,28). The inducible system used is a valuable tool to control AE1 expression in HEK293 cells, and the heterogeneous amounts of the protein at cell surface from one sample to the other actually validated our method based on the comparison of activities.…”

“…In the latter situation, affected individuals have an increase in RBC membrane permeability to cations and in most cases a reduction of anion movements through AE1. Expression of the mutated genes in Xenopus laevis oocytes induced abnormal Na ϩ and K ϩ fluxes (4,7,13,14,15,28,29). These data were consistent with the concept that the substitutions convert the protein from an anion exchanger into an unregulated cation channel, although, so far, no study has clearly demonstrated that the cation leaks are mediated through AE1.…”

“…11) or hereditary distal renal tubular acidosis (1). Recently, nine point mutations in the SLC4A1 gene (L687P, D705Y, R730C, S731P, H734R, E758K, R760Q, S762R, and G796R substitutions in the protein) have been reported (4,7,13,14,15,28,30). All these substitutions induce monovalent cation leaks in RBCs and are responsible for hereditary stomatocytosis (HSt) associated with hemolytic anemia (6).…”

Crystal structure of the anion exchanger domain of human erythrocyte band 3

“…3B). Both of these residues are conserved in AE1, and anion exchange is lost if either is mutated (19,(42)(43)(44). Though there is no apparent density for a substrate and substrate binding could be blocked by bound H2DIDS, a bicarbonate ion can be placed between the positive dipoles of TMs 3 and 10.…”